Methods of treatment

ABSTRACT

A method of producing active immunity against a bacterial or protozoal disease in a subject comprises administering to the subject a vaccine conjugate comprising a live bacteria or protozoa and a neutralizing factor bound to the live bacteria or protozoa. The neutralizing factor is selected from the group consisting of antibodies and antibody fragments. The live bacteria or protozoa is one capable of producing disease in the subject, and the antibody or antibody fragment is one capable of neutralizing the live bacteria or protozoa.

METHODS OF TREATMENT

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/027,084 filed Sep. 30, 1996.

FIELD OF THE INVENTION

[0002] The present invention relates to methods of producing activeimmunity against a bacterial or protozoal disease by administeringsubjects a vaccine conjugate, which conjugate is comprised of a livebacteria or protozoa and a neutralizing antibody or fragment thereof.

BACKGROUND OF THE INVENTION

[0003] Methods of producing active immunity against a viral disease byadministering a vaccine conjugate, the vaccine conjugate comprised of alive virus and a viral neutralizing antibody, are described in U.S. Pat.Nos. 5,397,568 and 5,397,569 to Whitfill et al. These references areconcerned with viral diseases only.

[0004] Methods of treating coccidiosis, a protozoan disease of bothbirds and mammals caused by various Eimieria species, are described inU.S. Pat. No. 4,935,007 to Baffundo et al. and U.S. Pat. No. 5,055,292to McDonald et al. In ovo inoculation against coccidiosis is describedin published PCT applications WO 96/40233 and 96/40234.

SUMMARY OF THE INVENTION

[0005] The present invention provides a method of producing activeimmunity against a bacterial or protozoal disease in a subject, themethod comprising administering to the subject a vaccine conjugatecomprised of a live bacteria or protozoa and a neutralizing factor boundto the live bacteria or protozoa. The neutralizing factor is selectedfrom the group consisting of antibodies and antibody fragments. Theantibody or antibody fragment is one capable of neutralizing the livebacteria or protozoa. The vaccine conjugate is administered in an amounteffective to produce an immune response to the live bacteria or protozoain the subject.

[0006] Another aspect of the present invention is a vaccine preparationuseful for producing active immunity against a bacterial or protozoaldisease in a subject. The vaccine preparation is a pharmaceuticallyacceptable formulation which comprises a vaccine conjugate. The vaccineconjugate comprises a live bacteria or protozoa and a neutralizingfactor bound to the live bacteria or protozoa. The neutralizing factoris selected from the group consisting of antibodies and antibodyfragments. The antibody or antibody fragment is capable of neutralizingthe live bacteria or protozoa. The vaccine conjugate is included in thepharmaceutically acceptable formulation in an amount effective toproduce an immune response to the live bacteria or protozoa in thesubject.

[0007] Another aspect of the present invention is an article ofmanufacture comprising a closed, pathogen-impermeable, container and asterile vaccine formulation as described above enclosed within thecontainer.

BRIEF DESCRIPTION OF DRAWINGS

[0008]FIG. 1 graphs the oocyst output in avians vaccinated with avaccine conjugate comprising 500 E. acervulina oocysts complexed witheither 2.5, 25 or 150 μl of polyclonal antibody, compared to anon-vaccinated control (cntrl) and a control vaccinated with oocysts butwithout antibody. Oocyst output is used as a measure of infectivity.

[0009]FIG. 2 graphs the oocyst output in avians vaccinated with avaccine conjugate comprising 500 E. acervulina oocysts complexed witheither 25 or 150 μl of polyclonal antibody, compared to a non-vaccinatedcontrol (cntrl) and a control vaccinated with oocysts but withoutantibody. Oocyst output is used as a measure of infectivity.

[0010]FIG. 3 graphs oocyst output after vaccination and low-dose E.acervulina challenge. Vaccination used a vaccine conjugate comprising500 E. acervulina oocysts complexed with either 2.5, 25 or 150 μl ofpolyclonal antibody; controls were a non-vaccinated control (cntrl) anda control vaccinated with oocysts but without antibody (0).

[0011]FIG. 4 graphs weight gain (in grams) in birds after vaccinationand a high-dose E. acervulina challenge. Vaccination used a vaccineconjugate comprising 500 E. acervulina oocysts complexed with either 25or 150 μl of polyclonal antibody; controls were a non-vaccinated control(cntrl) and a control vaccinated with oocysts but without antibody (0).

[0012]FIG. 5 graphs lesion scores in birds after vaccination and ahigh-dose E. acervulina challenge. Vaccination used a vaccine conjugatecomprising 500 E. acervulina oocysts complexed with either 25 or 150 μlof polyclonal antibody; controls were a non-vaccinated control (cntrl)and a control vaccinated with oocysts but without antibody (0).

DETAILED DESCRIPTION OF THE INVENTION

[0013] The present invention provides a vaccine preparation comprising alive organism (bacteria or protozoa) complexed with neutralizingantibodies specific to that organism. The amount of complexesneutralizing antibodies is such that the organism remains capable ofinducing an active immune response, while at the same time providingsome degree of protection against the deleterious effects of thepathogen. While applicants do not wish to be held to any single theory,it is currently believed that the present vaccine complex results insome form of delayed release of the pathogenic organism.

[0014] The present vaccine complex is thought to delay or initiallyprotect the vaccinated subject from the pathogenic effects of thevaccine organism. However, this delay or initial protection is onlytemporary (in contrast to what would be expected using a dead orinactivated vaccine organism). The vaccine organism in the complex doesultimately infect the subject, inducing an active immunity. The degreeof delay will be dependent on the amount of antibody used, theparticular vaccine organism, and the subject to be vaccinated. Such adelay in infection is important when vaccinating young subjects,particularly when large numbers of subjects are to be vaccinated. Forexample, it is easier and more cost-efficient to vaccinate chicks in ovocompared to vaccinating newly hatched chicks.

[0015] In a preferred embodiment of the present invention, theneutralizing factor is provided in an amount which delays the appearanceof pathological changes associated with infection of the subject by thelive vaccine organism. The “delay” is comparative; the pathologicalchanges are delayed in comparison to those which would occur if the livevaccine organism were administered without complexed neutralizingfactor.

[0016] Use of the present vaccine conjugates are safer than the use ofthe unconjugated organism yet are capable of inducing a protectiveactive immune response. The term “safe” is used herein to indicate thatthe benefits of vaccination outweigh any harm in the majority ofindividuals vaccinated.

[0017] Antibodies used in practicing the present invention are bacterialor protozoal neutralizing antibodies. Bacterial or protozoalneutralizing antibodies are those which combat the infectivity of abacteria or protozoa in vivo if the bacteria or protozoa and theantibodies are allowed to react together for a sufficient time. Thesource of the bacterial or protozoal neutralizing antibody is notcritical. They may originate from any animal, including birds (e.g.,chicken, turkey) and mammals (e.g., rat, rabbit, goat, horse). Thebacterial or protozoal neutralizing antibodies may be polyclonal ormonoclonal in origin. See. e.g. D. Yelton and M. Scharff, 68 AmericanScientist 510 (1980). The antibodies may be chimeric. See, e.g., M.Walker et al., 26 Molecular Immunology 403 (1989).

[0018] Bacterial or protozoal neutralizing antibodies used in practicingthe present invention may be immunoglobulins of any isotype, includingIgM, IgG, IgA, IgD, and IgE immunoglobulins. IgG and IgM are morepreferred, and IgG immunoglobulins (e.g., IgG1, IgG2, IgG3, IgG4) aremost preferred.

[0019] Antibody fragments used in practicing the present invention arefragments of bacterial or protozoal neutralizing antibodies which retainthe variable region binding site thereof. Exemplary are F(ab′)₂fragments, F(ab′) fragments, and Fab fragments. See generallyImmunology: Basic Processes, 95-97 (J. Bellanti Ed. 2d ed. 1985).

[0020] Antibodies or antibody fragments used in practicing the presentinvention may have additional elements joined thereto. For example, amicrosphere or microparticle may be joined to the antibody or antibodyfragment, as described in U.S. Pat. No. 4,493,825 to Platt, thedisclosure of which is incorporated herein by reference.

[0021] The present invention is particularly advantageously employedwith bacteria or protozoa which would be pathogenic (i.e., capable ofcausing disease) in the subject being treated if not for theirconjugation to the neutralizing factor. The pathogenicity of thebacteria or protozoa may be inherent in the bacteria or protozoa itselfor due to the susceptibility of the subject to be treated (e.g., birdsin ovo). In general, many pathogenic bacteria or protozoa have thepositive effect of evoking active immunity in subjects infectedtherewith, and many attenuated vaccine strains of bacteria or protozoahave the capability of causing at least some disease in subjects. Hence,the term “pathogenic,” as used to describe bacteria or protozoa herein,means that the harm caused to subjects by administration of the bacteriaor protozoa outweighs any benefit which would result therefrom. An“active” or “live” organism refers to one which is not killed. A“vaccine organism” refers to one which is used for the induction ofprotective immune response, even though negative side effects may occur(in such cases the benefit of the active immunity outweighs any negativeside effects). It is preferred that the bacteria or protozoa be a liveorganism one capable of producing an active immune response thereto inthe subject being treated.

[0022] The vaccine conjugate is included in the vaccine formulations inan amount per unit dose sufficient to evoke an active immune response tothe bacteria or protozoa in the subject to be treated. The term “immuneresponse,” as used herein, means any level of protection from subsequentexposure to the bacteria or protozoa which is of some benefit in apopulation of subjects, whether in the form of decreased mortality,decreased lesion scores, improved feed conversion ratios, or thereduction of any other detrimental effect of the disease, regardless ofwhether the protection is partial or complete.

[0023] With respect to the degree of protection provided by theneutralizing factor, the quantity of the neutralizing factoradministered in combination with the bacteria or protozoa in the vaccineneed not be sufficient to provide complete protection from the bacteriaor protozoa, as long as the detrimental response produced by thebacteria or protozoa is reduced to a level at which the benefits of theimmune response produced outweigh any harm resulting from the infection.

[0024] The term “subjects,” as used herein, is intended to include,among other things, both mammals and birds. Exemplary mammals includemice, rats, pigs, rabbits, sheep, ferrets, dogs, cats, cows, horses andprimates, including man. The term “bird” is intended to include males orfemales of any avian species, but is primarily intended to encompasspoultry which are commercially raised for eggs or meat. Accordingly, theterm “bird” is particularly intended to encompass hens, cocks and drakesof chickens, turkeys, ducks, geese, quail and pheasant.

[0025] Bacteria that may be used in carrying out the present inventioninclude, but are not limited to, Actinobacillosis lignieresi,Actinomyces bovis, Aerobacter aerogenes, Anaplasma marginale, Bacillusanthracis, Borrelia anserina; Brucella canis, Clostridium chauvoei, C.hemolyticium C. novyi, C perfringens, C. septicum, C. tetani,Corynebacterium equi, C. pyogenes, C. renale, Cowdria ruminantium,Dermatophilus congolensis, Erysipelothrix insidiosa, Escherichia coli,Fusiformis necrophorus, Haemobartonella canis, Hemophilus spp. H. suis,Leptospira spp., Moraxella bovis, Mycoplasma spp. M hyopneumoniae,Nanophyetus salmincola, Pasteurella anatipestifer, P. hemolytica, P.multocida, Salmonella abortus-ovis, Shigella equirulis, Staphylococcusaureus, S. hyicus. S. hyos, Streptococcus agalactiae, S. dysgalactiae,S. equi, S. uberis, and Vibrio fetus (for the corresponding diseases,see Veterinary Pharmacology and Therapeutics 5th Edition, pg 746 Table50.2 (N. Booth and L. McDonald Eds., 1982)(Iowa State University Press);and Corynebacterium diptheriae, Mycobacterium bovis, M. leprae, Mtuberculosis, Nocardia asteroides, Bacillus anthracis, Clostridiumbotulinum, C. difficile, C. perfringens, C. tetani, Staphylococcusaureus, Streptococcus pneumoniae, S. pyogenes, Bordetella pertusiss,Psudomonas aeruginos, Campylobacter jejuni, Brucella spp., Francisellatularenssis, Legionella pneumophila, Chlamydia psittaci. C. trachomatis,Escherichia coli, Klebsiella pneumoniae, Salmonella typhi, S.typhimurium, Yersinia enterocolitica, Y pestis, Vibrio cholerae,Haemophilus influenza, Mycoplasma pneumoniae, Neiseseria gonorrhoeae, Nmeninigitidis, Coxiella burneti, Rickettsia mooseria, R. prowazekii, R.rickettsii, R. tsutsugamushi, Borrelia spp., Leptospira interrogans,Treponema pallidum, and Listeria monocytogenes (for the correspondingdiseases see R. Stanier et al., The Microbial World, pg. 637-38 Table32.3 (5th Edition 1986).

[0026] Protozoa that may be used in carrying out the present inventioninclude, but are not limited to, the coccidiosis-causing Eimeria species(E. tenella, E. necatrix, E. brunetti, E. acervulina, E. mivati, and E.maxima), Anaplasma marginale, Giardia species (e.g., Giardia lamblia),Babesia species (e.g., B. canis, B. gibsoni, B. equi, B. caballi, B.bigemina, B. argentina, B. divergens, and B. bovis) Trichomonas foetus,Entamoeba histolytica, and Balantidium coli; Plasmodium species (e.g.,P. falciparum, P. malariae, P. vivax, and P. ovale), Leishmania species(e.g., L. donovani, L. braziliensis, L. tropica, and L. mexicana),Trypanosoma species e.g., T brucei and T. cruzi), Entamoeba histolytica,Trichomonas vaginalis, Toxoplasmosa gondii, and Pneumocystis carinii. Asused herein, an “avian protozoan” is one known to infect avians.

[0027] The organisms may be administered in any suitable form, includingspores or cysts thereof For example, infective coccidial organisms maybe administered in the form of sporulated oocysts, sporozoites, andsporocysts.

[0028] The exact number of the organisms to be administered in the formof a conjugate is not critical except that the number must be effectiveto engender an immunological response by the animal. In general,depending on the organism administered, the site and manner ofadministration, the age and condition of the subject, etc., the numberof the organisms will range from 1, 10, or 100 organisms up to 1,000,10,000, 100,000 or 1 million organisms. Where the organisms areadministered as a conjugate to birds in ovo (within eggs), the dosagemay be from 50, 100, or 500 up to 2,000, 10,000, 20,000, 30,000, 50,000or 100,000 organisms or more.

[0029] Subjects may be administered vaccines of the present invention byany suitable means. Exemplary are by oral administration, byintramuscular injection, by subcutaneous injection, by intravenousinjection, by intraperitoneal injection, by eye drop or by nasal spray.When the subject to be treated is a bird, the bird may be a hatchedbird, including a newly hatched (i.e., about the first three days afterhatch), adolescent, and adult birds. Birds may be administered thevaccine in ovo, as described in U.S. Pat. No. 4,458,630 to Sharma (thedisclosure of this and all other patent references cited herein is to beincorporated herein by reference).

[0030] The in ovo administration of the vaccine involves theadministration of the vaccine to eggs. Eggs administered the vaccine ofthe present invention are fertile eggs which are preferably in thefourth quarter of incubation. Chicken eggs are treated on about thefifteenth to nineteenth day of incubation, and are most preferablytreated on about the eighteenth day of incubation (the eighteenth day ofembryonic development). Turkey eggs are preferably treated on about thetwenty-first to twenty-sixth day of incubation, and are most preferablytreated on about the twenty-fifth day of incubation.

[0031] Eggs may be administered the vaccine of the invention by anymeans which transports the compound through the shell. The preferredmethod of administration is, however, by injection. The site ofinjection is preferably within either the region defined by the amnion,including the amniotic fluid and the embryo itself, in the yolk sac, orin the air cell. Most preferably, injection is made into the regiondefined by the amnion. By the beginning of the fourth quarter ofincubation, the amnion is sufficiently enlarged that penetration thereofis assured nearly all of the time when the injection is made from thecenter of the large end of the egg along the longitudinal axis.

[0032] The mechanism of egg injection is not critical, but it ispreferred that the method not unduly damage the tissues and organs ofthe embryo or the extraembryonic membranes surrounding it so that thetreatment will not decrease hatch rate. A hypodermic syringe fitted witha needle of about 18 to 22 gauge is suitable for the purpose. To injectinto the air cell, the needle need only be inserted into the egg byabout two millimeters. A one inch needle, when fully inserted from thecenter of the large end of the egg, will penetrate the shell, the outerand inner shell membranes enclosing the air cell, and the amnion.Depending on the precise stage of development and position of theembryo, a needle of this length will terminate either in the fluid abovethe chick or in the chick itself. A pilot hole may be punched or drilledthrough the shell prior to insertion of the needle to prevent damagingor dulling of the needle. If desired, the egg can be sealed with asubstantially bacteria-impermeable sealing material such as wax or thelike to prevent subsequent entry of undesirable bacteria.

[0033] It is envisioned that a high speed automated egg injection systemfor avian embryos will be particularly suitable for practicing thepresent invention. Numerous such devices are available, exemplary beingthose disclosed in U.S. Pat. No. 4,681,063 to Hebrank and U.S. Pat. Nos.4,040,388, 4,469,047, and 4,593,646 to Miller. All such devices, asadapted for practicing the present invention, comprise an injectorcontaining the vaccine described herein, with the injector positioned toinject an egg carried by the apparatus with the vaccine. Other featuresof the apparatus are discussed above. In addition, if desired, a sealingapparatus operatively associated with the injection apparatus may beprovided for sealing the hole in the egg after injection thereof.

[0034] Preferred egg injection apparatus for practicing the presentinvention is disclosed in U.S. Pat. Nos. 4,681,063 and 4,903,635 toHebrank, the disclosures of which are incorporated herein by reference.This device comprises an injection apparatus for delivering fluidsubstances into a plurality of eggs and suction apparatus whichsimultaneously engages and lifts a plurality of individual eggs fromtheir upwardly facing portions and cooperates with the injection meansfor injecting the eggs while the eggs are engaged by the suctionapparatus. The features of this apparatus may be combined with thefeatures of the apparatus described above for practicing the presentinvention. Preferred subjects for carrying out the present invention arebirds.

[0035] The method of the present invention is preferably carried out onbirds in ovo.

[0036] A vaccine conjugate of the present invention is made by mixingthe neutralizing factor with a live bacteria or protozoa in apharmaceutically acceptable carrier for a time sufficient to form a livebacteria or protozoa-neutralizing factor conjugate (for example, bycombining the neutralizing factor and bacteria or protozoa in a commonliquid carrier prior to administration to a subject, until a conjugateis formed). This can advantageously be carried out by simply addinghyperimmune sera containing neutralizing antibodies to an aqueoussolution containing the live bacteria or protozoa. Vaccine formulationsof the present invention preferably comprise the vaccine conjugate inlyophilized form or the vaccine conjugate in a pharmaceuticallyacceptable carrier. Pharmaceutically acceptable carriers are preferablyliquid, particularly aqueous, carriers. For the purpose of preparingsuch vaccine formulations, the neutralizing factor and bacteria orprotozoa may be mixed in sodium phosphate-buffered saline (pH 7.4),conventional media such as MEM, or bacterila growth medium. The vaccineformulation may be stored in a sterile glass container sealed with arubber stopper through which liquids may be injected and formulationwithdrawn by syringe.

[0037] The vaccine conjugate or complex of the present invention is acomplex or conjugate of antibodies and live vaccine organisms; the bondbetween antibody and vaccine organism is a releasable bond and is not acovalent bond. The amount of neutralizing antibodies suitable for usewith a given vaccine organism and a given subject can be readilydetermined using techniques available in the art. Use of too littleantibody will result in undesirably early or severe pathogenic effectscaused by the vaccine organism; use of too much antibody may inactivatethe vaccine organism completely or render it incapable of inducing aprotective immune response.

[0038] Vaccine formulations of the present invention may optionallycontain one or more adjuvants. Any suitable adjuvant can be used,including chemical and polypeptide immunostimulants which enhance theimmune system's response to antigens. Preferably, adjuvants such asaluminum hydroxide, aluminum phosphate, plant and animal oils, and thelike are administered with the vaccine conjugate in an amount sufficientto enhance the immune response of the subject to the vaccine conjugate.The amount of adjuvant added to the vaccine conjugate will varydepending on the nature of the adjuvant, generally ranging from about0.1 to about 100 times the weight of the bacteria or protozoa,preferably from about 1 to about 10 times the weight of the bacteria orprotozoa.

[0039] The vaccine formulations of the present invention may optionallycontain one or more stabilizer. Any suitable stabilizer can be used,including carbohydrates such as sorbitol, manitol, starch, sucrose,dextrin, or glucose; proteins such as albumin or casein; and bufferssuch as alkaline metal phosphate and the like. The use of a stabilizeris particularly advantageous when the vaccine formulation is alyophilized formulation.

[0040] The present invention is explained in greater detail in thefollowing non-limiting Examples.

EXAMPLE 1 Bacterial Species

[0041] The bacterium Pasteurella multocida causes an acute highlycontagious disease in many avian species. The disease, Fowl cholera,often occurs as a septicemic disease resulting in high morbidity andmortality. There are several live vaccines for Fowl cholera that can beadministered to both chickens and turkeys.

[0042] A strain of P. multocida is complexed in vitro with antibodiesspecific to P. multocida to form bacterium-antibody complexes. Differentratios of bacterium to antibody are tested to determine a ratio thatdoes not completely inactivate the bacterium, and still allows an activeimmune response to occur. These complexes are used as a vaccine ineither chickens or turkeys. The responses of the vaccinate are followedand compared to the responses of birds vaccinated with the same dose ofP. multocida vaccine not complexed with antibody. Lesions followingvaccination, antibody response over time and general bird healty aremonitored throughout the trial.

[0043] Other bacteria are also tested in a similar manner. Thesebacteria include, but are not limited to, Mycoplasma gallisepticum inchickens or turkeys, Bordetella avium in turkeys, Salmonella species inchickens or turkeys, and Salmonella and Listeria species in rodents.

[0044] Vaccine conjugates are administered to birds either in ovo asdescribed above or after hatch.

EXAMPLE 2 Protozoal Species

[0045] The protozoan Eimeria acervulina (specifically, sporocysts and/oroocysts thereof) is complexed in vitro to antibodies specific to Eimeriaacervulina to form protozoan-antibody complexes. These complexes areused as vaccines in chickens. The responses of the protozoan-antibodycomplex vaccinates are followed and compared to the responses of birdsvaccinated with the same dose of E acervulina not complexed withantibody. Different ratios of protozoan to antibody are tested todeterimine a ratio that does not completely inactivate the protozoan,and still allows an active immune response to occur. Oocyst output invaccinates feces, intestinal absorptive ability (assessed by cartenoiduptake) and body weight are determined post vaccination. At 10-21 dayspost vaccination birds in each vaccinated group are given a virulentchallenge with E. acervulina Oocyst output in vaccinates feces, bodyweight gain during the challenge period and intestinal absorptiveability (assessed by cartenoid uptake) are determined for vaccinates andnon-vaccinated controls.

[0046] Other Eimeria species are also tested in chickens, turkeys orrodents, as well as Cryptosporidium species in chickens or turkeys andHistomonas meleagridis in chickens or turkeys.

[0047] Vaccine conjugates are administered to birds either in ovo asdescribed above or after hatch.

EXAMPLE 3 Eimeria Oocyst Output Following Vaccination

[0048] Chickens were vaccinated and studied to determine the effects ofcomplexing an E. acervulina oocyst vaccine with antibody.

[0049] Four vaccination strategies were studied. Treatment groups werevaccinated (by oral gavage) with 500 E. acervulina sporulated oocystscomplexed with either 0, 2.5, 25 or 150 units of polyclonal antibodyspecific for E. acervulina. Each treatment was provided to three groupsof five Leghorn chickens (15 birds total in each treatment group; 60treatment birds overall). A control group of fifteen birds (5 birds inthree repetitions) received no oocysts and no antibody, but were treatedwith oral gavage of 0.1 ml PBS administered on the day of hatch and weresubsequently challenged.

[0050] Units of antibody is defined on a volumetric basis, where 1 μl=1unit. An ELISA assay has been used to determine the “titer units” of theantibody preparation used in the present example; however, this assayhas not been validated. By the ELISA assay, the E. acervulina antibodypreparation had a titer of 90,782 units/ml. The doses used hereinprovide relative comparisons; the appropriate amount of antibody to becomplexed with a given organism will depend on the organism, theantibody preparation, and the intended subject. One skilled in the art,using techniques known in the art, would be able to determineappropriate organism:antibody ratios for a given usage.

[0051] The oocysts and antibodies were mixed together in PBS for aminimum of one hour prior to vaccination at room temperature. Vaccinecomplex was then stored at 4 C. until administration. Chickens werevaccinated on the day of hatch.

[0052] Oocyst output following vaccination was determined by collectingfeces on days 4 to 8 post vaccination, and counting oocyst in the feces.The mean and standard deviation of oocyst output was determined for eachgroup. As shown in Table 1, use of 150 μl of antibodies complexed with500 oocysts significantly reduced oocyst output. TABLE 1Infectivity-Oocyst Output per Bird Vaccination Average Oocysts/BirdTreatment (x 10⁶) None Mean 0.00 Standard Deviation 0.00 N 3² 0 μl Abs +oocysts (a)¹ Mean 11.65 Standard Deviation 2.91 N 3² 2.5 μl Abs +oocysts (a)¹ Mean 10.18 Standard Deviation 2.54 N 3² 25 μl Abs + oocysts(a)¹ Mean 10.61 Standard Deviation 4.28 N 3² 150 μl Abs + oocysts (b)¹Mean 5.40 Standard Deviation 1.94 N 3²

EXAMPLE 4 Eimeria Oocyst Output Following Challenge

[0053] Birds in the treatment groups described in Example 1 were thenchallenged on Day 13 posthatch with 250 oocysts of E. acervulina in PBSadministered by oral gavage. Feces was collected on days four to eightpost-challenge, and average oocyst output was determined as a percentageof the oocyst output of the control group (the statistical modelincluded the control group). Results are provided in Table 2. Greateroutput of oocysts following challenge indicates less protection againstthe pathogen challenge. TABLE 2 Protection after Challenge AverageOocyst Output per Bird; Average Vaccination Post-challenge OocystsTreatment (x 10⁶) (% of control)² Control² A¹ A¹ Mean 23.08 100.00Standard Deviation 1.67 7.22 N 3 3 0 μl Abs + oocysts C¹ C¹ Mean 5.2022.54 Standard Deviation 0.72 3.13 N 3 3 2.5 μl Abs + oocysts B¹ C¹ Mean8.72 37.76 Standard Deviation 2.45 10.60 N 3 3 25 μl Abs + oocysts B¹ B¹Mean 9.76 42.29 Standard Deviation 1.78 7.70 N 3 3 150 μl Abs + oocystsB¹ B¹ Mean 10.49 45.47 Standard Deviation 2.33 10.10 N 3 3

EXAMPLE 5 Protection Following Challenge

[0054] Data provided in Table 2 was re-analyzed without including thecontrol group data in the statistical model. Results are provided inTable 3. TABLE 3 Average Oocysts Average Vaccination Post-challengeOocysts Treatment (x 10⁶) (% of control) Control² Mean 23.08 100.00Standard Deviation 1.67 7.22 N 3 3 0 μl Abs + oocysts B¹ B¹ Mean 5.2022.54 Standard Deviation 0.72 3.13 N 3 3 2.5 μl Abs + oocysts AB¹ AB¹Mean 8.72 37.76 Standard Deviation 2.45 10.60 N 3 3 25 μl Abs + oocystsA¹ A¹ Mean 9.76 42.29 Standard Deviation 1.78 7.70 N 3 3 150 μl Abs +oocysts A¹ A¹ Mean 10.49 45.47 Standard Deviation 2.33 10.10 N 3 3

[0055] The results provided in Examples 1-3 indicate that use of 150 μlantibody in conjunction with the vaccination dose of 500 E acervulinaoocysts resulted in either a lessening of the pathogenic effects of thevaccination (compared to use of same vaccination with lesser amounts ofantibody, or no antibody; indicated by decreased oocysts output aftervaccination), or possibly a delay in the pathogenic effects of thevaccination dose. As shown in Table 1, the use of 150 μl of antibodycomplexed to the vaccine oocysts resulted in a lower infectivity levelthan vaccination without antibodies or the use of lesser amounts ofantibodies (p≦0.15).

[0056] As shown in Table 2, birds vaccinated with antibody-oocystvaccine had reduced oocyst output after challenge with 250 E. acervulinaoocysts, compared to unvaccinated birds. These results indicate that theantibody-oocyst vaccine preparation is effective in inducing aprotective immune response against the challenging pathogen. Protectionwas highest in the treatment group vaccinated with oocysts but withoutany antibody, with a significance level of 0.05. As shown in Table 3,among birds treated with antibody-oocyst vaccine, the highest protectionis again seen in the group treated with oocyst but no antibody.

[0057] The above results indicate that use of a vaccine complex ofantibody and oocyst resulted in either a lessening of the pathogeniceffects of the vaccine oocysts, or a delay in the pathogenic effects ofthe vaccine oocysts, while still engendering a protective immuneresponse. Either effect would be expected to increase the safety of avaccine, either by allowing administration of the vaccine to subjectswho are more susceptible to the pathogenic effects of the vaccineorganism, or to subjects at a younger age (such as to avians in ovo).

[0058] The foregoing are illustrative of the present invention, and arenot to be taken as limiting thereof. The invention is defined by thefollowing claims, with equivalents of the claims to be included therein.

EXAMPLE 6 Use of Antibody-Oocyst (Eimeria acervulina) Vaccine Conjugatein Low-Dose Challenge Model

[0059] This study tested a 500 Eimeria acervulina oocyst vaccinecomplexed with varying amounts of antibody (2.5 to 150 μl). Treatmentswere compared to a non-vaccinated control, and a control vaccinatedwithout antibodies. All treatments were administered on the day ofhatch. Oocyst output after vaccination on Days 4-8 was measured for alltreatments. Oocyst output was also measured after a Day 13 low dosechallenge.

[0060] Materials and Methods: Hyvac SPF leghorns were used to rule outany effect of maternal antibodies. Polyclonal antibody was produced fromchickens immunized with E. acervulina oocysts. Two antibody preparationswere combined to create an E. acervulina antibody with a final titer of90,782. Treatment groups and experimental design are shown in Table 4.TABLE 4 Total # Antibody # of Birds/ of Treatment Oocyst Dose (μl) Rep #Reps Birds 1 (A, B, C)  0 0 5 3 15 2 (A, B, C) 500 0 5 3 15 3 (A, B, C)500 2.5 5 3 15 4 (A, B, C) 500 25 5 3 15 5 (A, B, C) 500 150 5 3 15

[0061] The vaccine complex was produced by mixing oocysts (USDA #12 Lot28-131-36) with antibody in the appropriate volume. The complex wasincubated at ambient temperature for one hour before administration.Birds were gavaged on Day of Hatch with a 200 μl dose of the respectivetreatment. Fecal material was collected form Day 4 to Day 8. Fecalsamples were processed and counted using McMaster's chambers todetermine oocyst output per bird.

[0062] Birds were moved to a brooder unit and challenged on Day 13post-hatch with a low dose challenge (250 E. acervulina oocysts). Feceswas collected Days 4-8 post challenge and enumerated as described above.

[0063] The vaccinated control showed ≅12×10⁶ oocyst output per bird. The2.5 μl and 25 μl antibody treatments showed similar results, however,the 150 μl antibody treatment may have had an inhibitory effect onoocyst output, having only 46% of the output compared to the control.See FIG. 1.

[0064] After a low dose challenge, similar results were seen in all theantibody vaccinated treatment groups. FIG. 3. The three antibodytreatment groups averaged approximately 40% output of control. Thevaccinated control exhibited an output of only 23% of control.

EXAMPLE 7 Use of Antibody-Oocyst (Eimeria acervulina) Vaccine Conjugatein High-Dose Challenge Model

[0065] Two antibody-oocyst vaccine conjugate formulations were tested ina high-dose challenge model. Infectivity was measured by oocyst outputand response to challenge was measured by weight gain and lesion scores.The vaccine conjugates consisted of 500 E. acervulina oocysts complexedwith either 25 or 150 μl of antibody (as described in Example 6); seeTable 5. The same bird strain, antibody, and oocyst lot was used as inExample 6 above. TABLE 5 Total # Antibody # of Birds/ Of Treatment #Oocyst Dose (μl) Rep # Reps Birds 1 (A, B, C)  0  0 10 3 30 2 (A, B, C)500  0 10 3 30 3 (A, B, C) 500 25 10 3 30 4 (A, B, C) 500 150  10 3 30

[0066] Vaccines were prepared as described above and administered on Day0 post-hatch in 200 μl volume. Fecal material was collected from Day 4-8and enumerated.

[0067] Post-challenge parameters measured in the present experimentdiffered from Example 6. A high dose challenge was administered to alltreatment groups on Day 13 and weights of each individual bird wererecorded. After eight days (Day 21) the birds were weighed and thelesions scored.

[0068] Oocyst output was lower in this experiment for the vaccinatedcontrol, as well as for the two antibody treatments, compared to Example6; the vaccinated control (no antibody) showed a 20-fold decrease inoocyst output (see FIG. 2). The cause of this reduction is not clear.

[0069] A high dose challenge (500 oocyst challenge) was administered toall the treatment groups and weight gain and lesion scores wereexamined. Weight gain results (FIG. 4) did not show a difference amongthe treatment groups, including vaccinated and non-vaccinated controls.Lesion score data (FIG. 5) indicated protection of all vaccinated groupsover the non-vaccinated control.

EXAMPLE 8 Pasteurella multocida

[0070] Production of antiserum to P. multocida: Ten SPF chickens werehoused in a clean room from hatch; at four weeks of age each birdreceived (subcutaneous injection in the neck) 0.5 mls of Solvay's“Pabac”, a commercial inactivated P. multocida oil emulsion vaccinecontaining serotypes 1, 3 and 4; at eight weeks of age, an additional0.5 mls was injected subcutaneously into the neck and another 0.5 mlswas injected intramuscularly in the right breast. Approximately 20 mlsof blood was removed from each bird by cardiac puncture at ten weeks ofage. Antiserum was collected from the blood, pooled, and filteredthrough a 0.45 μm filter. After numerous sterility tests.all producednegative results, the antisera was placed into different sized vials andplaced in a −20 degree C. freezer until use.

Isolation and titer determination of the Cu and M9 strains of P.multocida

[0071] The Cu and M9 strains of P. multocida were grown from livevaccines, Choleramune Cu and Multimune M respectively, each produced byBiomune. It was determined that these strains of P. multocida freezebest at −70 degrees C. in a mixture of 90% culture and 10% glycerol.

EXAMPLE 9 Hatchability of Eggs Inoculated at Day 18 with P. multocida

[0072] This experiment was designed to determine if different numbers ofcolony forming units (CFUs) of P. multocida strains Cu and M9 affect thehatchability of SPF eggs following in ovo inoculation at day 18 ofincubation. Each strain of P. multocida was diluted in PhosphateBuffered Saline (PBS) to produce three dilutions: 1000 CFUs per 0.1 ml;100 CFUs per 0.1 ml; and 10 CFUs per 0.1 ml. At day 18 of incubation,0.1 mls of each dilution for each strain was inoculated into fourteenSPF eggs. Thirteen eggs were inoculated with 0.1 ml of a mixture ofBrain Heart Infusion Broth (BHI), PBS, and Glycerol (vehicle control)and thirteen eggs received no inoculation.

[0073] The results in Table 6 show that hatchability of SPF eggs wasseverely depressed in groups 2 through 7. TABLE 6 Hatchability of SPFEggs Following Inoculation at Day 18 with P. multocida Group 5 Group 6Group 8 Group 1 Group 2 Group 3 Group 4 M9, M9 Group 7 No BHI, PBS Cu,1000 Cu, Cu, 1000 100 M9, Inocu- Glycerol CFUs 100 CFUs 10 CFUs CFUsCFUs 10 CFUs lation Normal 12  3  1 11 Hatched Un-  1 14 14 11 14 14 13 2 Hatched % 92  0  0 21  0  0  7 85 Hatched

[0074] Each inoculated egg was inoculated with 0.1 ml of a culture/PBSmixture containing the appropriate number of CFUs. The dilutionscontaining 1×10(2) CFUs/ml were titered for each strain and the titersdiffered somewhat from the target numbers. Group 4 actually contained10.5 CFUs/egg, Group 3 contained 105 CFUs/egg, and Group 2 contained1050 CFUs/egg. Group 7 contained 12.5 CFUs/egg, Group 6 contained 125CFUs/egg, and Group 5 contained 1250 CFUs/egg. Group 1 eggs wereinoculated with 0.1 ml of a BHI/PBS/Glycerol mixture.

EXAMPLE 10 Colony Growth of P. multocida after Complexing with ChickenAntiserum

[0075] One ml of chicken P. multocida antiserum (see Example 8) wasremoved from −20 degrees C. and thawed at room temperature. The serumwas serially diluted, 2-fold, in PBS. One half ml of each serum dilutionwas mixed with 0.5 ml of P. multocida Cu strain culture containing 100CFUs/0.5 ml. This mixture reacted together for one hour at a roomtemperature. When one hour had elapsed, 0.5 ml of each serum dilutionmixture was plated on TSA plates in duplicate and the 200 CFUs/ml P.multocida Cu strain stock solution was plated in triplicate. The plateswere incubated for 48 hours at 37 degrees C. Colonies were counted after24 hours and 48 hours of incubation. Results are shown in Tables 7 and8. TABLE 7 48 Hours 24 Hours change in CFUs/1.0 #CFUs CFU count/ Serumml ex- 1.0 ml of Dilution CFUs/0.5 ml Avg of mixture pected* mixture1:16 114 + 96  = 105 × 2 = 210 152 no change 1:32 72 + 75 = 73.5 × 2 =147 152 no change 1:64 92 + 86 = 89 × 2 = 178 152 no change 1:128 98 +71 = 84.5 × 2 = 169 152 no change 1:256 81 + 92 = 86.5 × 2 = 173 152 nochange 1:512 64 + 80 = 72 × 2 = 144 152 no change 1:1024  63 + 101 = 82× 2 = 164 152 no change 1:2048 73 + 73 = 73 × 2 = 146 152 no change1:4096 68 + 81 = 74.5 × 2 = 149 152 no change 1:8192 60 + 61 = 60.5 × 2= 121 152 no change 1:16384 42 + 61 = 51.5 × 2 = 103 152 +1 colony1:32768 56 + 59 = 57.5 × 2 = 115 152 no change

[0076] TABLE 8 Titer of the 2 × 10(2) Stock Solution of P. multocida CuStrain CFU/0.5 ml Average Per 1.0 ml 176 152 × 2 = 304 131 150

[0077] Stock solution samples in Table 8 were plated after all mixtureswere prepared. The solution did not sit for an additional hour. Amixture of 0.5 ml of this stock solution and 0.5 ml of antiserum yieldsan expected 152 CFUs/ml of mixture.

[0078] The results show a decrease in the number of bacterial CFUs fromthe least dilute antiserum to the most dilute antiserum (Table 7). Thismay be due to reaction time. The most dilute antiserum reacted longerwith the live culture than the least dilute antiserum (20-30 minuteslonger). The increased time may have lead to greater numbers of coloniesdying off. Repeated vortexing of the stock solution (3-4 CFUs/ml) mayhave also contributed to the observed decrease in colony count. The nextExample investigates this trend further.

EXAMPLE 11

[0079] This experiment investigated the reaction between colonies of P.multocida Cu strain and the same dilutions of the P. multocida antiserumas shown in Table 6. Instead of vortexing the “2×10(2)” stock solutionof P. multocida Cu strain continually, as in the previous experiment,the solution was gently mixed by pipetting. To form eachantibody-bacterium complex, one ml of antiserum dilution was mixed withone ml of stock culture dilution. This mixture was allowed to react forfour hours. The stock solution was plated on TSA plates in triplicateand was found to contain 186 CFUs/ml (Table 9). The remainder of thissolution was also allowed to remain at room temperature for four hours.

[0080] Following the four hour reaction time, 0.5 ml of eachantibody-bacterium mixture was mixed and plated in duplicate on TSA. The186 CFU/ml stock solution was mixed and plated on TSA in triplicate (0.5ml/plate). Colonies were counted at 24 hours after incubation at 37degrees C. and again after 96 hours. The results are provided in Tables9 and 10. TABLE 9 Titer of the planned 2 × 10(2) CFUs/ml final stocksolution of P. multocida strain Cu Immediately after After sitting atroom temperature Adding 1 ml samples to serum for 4 hours CFU/0.5 mlAverage Per 1 ml CFU/0.5 ml Average Per 1 ml 39 93 93 186 47 44.7 89 48

[0081] TABLE 10 24 Hours Serum #CFUs Dilution CFUs/0.5 ml Avg CFUs/1.0ml expected* 96 Hours 1:16 114 + 92  103 206 93 no change 1:32 79 + 7376 152 93 no change 1:64 50 + 48 49  98 93 no change 1:128 58 + 69 63.5127 93 no change 1:256 52 + 67 59.5 119 93 no change 1:512 72 + 63 67.5135 93 no change 1:1024 55 + 63 59 118 93 no change 1:2048 55 + 62 58.5117 93 no change 1:4096 51 + 53 52 104 93 no change 1:8192 28 + 34 31 64 93 no change 1:16384 20 + 26 23  46 93 no change 1:32768 24 + 20 22 44 93 no change

[0082] The data in Table 10 shows the same decreasing colony count withincreasing antiserum dilution as was seen in the previous experiment,slightly more pronounced. There appears to be more CFUs/ml than expectedin dilution mixtures 1:16 through 1:4096. Similar findings were alsopresented in Table 7. The colony counts found in Table 9 suggest theCFUs die off in PBS over time in the absence of serum. This may accountfor the lower than expected colony counts in dilutions 1:8192 through1:32768 in both studies. The antiserum may be demonstrating some growthinhibiting capabilities at the higher dilutions while exhibiting growthenhancement effects in the least dilute mixtures. The next experimentwas conducted to investigate possible causes of these observations.

EXAMPLE 12

[0083] The effects of P. multocida antiserum and negative serum (chickenserum containing no P. multocida antibodies) on the growth of the M9strain of P. multocida was compared in vitro. Serum dilutions and P.multocida culture dilutions were prepared as in Example 11. One ml ofculture containing 139 CFUs per ml (Table 6) was mixed with one ml ofdilute serum. Three dilutions (1:16, 1:512, and 1:32768) of negativeserum were prepared and mixed with the appropriate amount of bacterialculture. The bacterial culture/serum dilution mixtures reacted togetherfor one hour at room temperature.

[0084] The serum antibody-bacterium mixtures were plated on TSA plates(0.5 ml) in duplicate and the negative serum bacterium mixtures wereplated in triplicate following the one hour reaction time. The P.multocida strain M9 stock solution was plated in triplicate after adding1.0 ml to all antiserum and negative serum dilutions and again afterallowing it to remain at room temperature for one hour. Colonies werecounted after incubating for 24 hours at 37 degrees C. and any changesin counts were noted after 48 hours of incubation. The results of thesecounts are given in Tables 11 and 12. TABLE 11 Titer of the planned 2 ×10(2) CFU/ml final stock solution of P. multocida M9 Immediately afterAfter sitting at room temperature adding 1 ml samples to serum for 1hour CFU/0.5 ml Average Per 1 ml CFU/0.5 ml Average Per 1 ml 68 61 7669.3 138.6 63 64.3 128.6 64 69

[0085] TABLE 12 P. multocida antiserum Negative antiserum after 24 hrs #CFUs after 24 hours Serum CFUs/ Per expected CFUs/ Per Dilution 0.5 mlAvg. 1 ml in 1 ml* 0.5 ml Avg. 1 ml 1:16 36 + 34 35 70 69 34 + 55 + 5046.3 92.6 1:32 39 + 33 36 72 69 1:64 40 + 37 38.5 77 69 1:128 53 + 33 4386 69 1:256 33 + 35 34 68 69 1:512 34 + 51 42.5 85 69 44 + 43 + 40 42.384.6 1:1024 36 + 35 35.5 71 69 1:2048 32 + 37 34.5 69 69 1:4096 34 + 2931.5 63 69 1:8192 34 + 44 39 78 69 1:16384 24 + 25 24.5 49 69 1:3276818 + 38 28 56 69 40 + 39 + 33 37.3 74.6

[0086] There were no changes in the colony counts after 48 hours ofincubation at 37 degrees C.

[0087] The data in Examples 10-12 show initial increases leading togradual decreases in the number of CFUs as antiserum to P. multocidabecame more dilute. The combined data suggest that both strains of P.multocida may actually use the serum as a growth medium. As the serumconcentration decreases so does the growth of P. multocida. Some of theresults suggest that P. multocida dies off when diluted in PBS and sitsat room temperature for between one and four hours. This may account forthe fewer than expected CFUs observed from the 1:8192 dilutions through1:32768 dilutions. As the serum becomes less concentrated, the PBSconcentration increases. There was not a large difference, though, inthe stock colony counts in the present example, before and after a onehour waiting period, yet the low counts in the higher dilutions stillexisted.

[0088] The colony counts for the serum mixtures containing no antibodiesto P. multocida, when compared to their P. multocida antibody-containingcounterparts and stock counts, may be showing some growth inhibitioncapabilities of the antiserum with P. multocida antibodies. CFU countsin the samples without P. multocida antiserum started high and decreasedbut not to a level lower than expected when compared to the stock CFUcounts after one hour. CFU counts were lower in two of three samplescontaining antibodies to P. multocida than in the negative serum sampleswithout P. multocida antibodies. A growth inhibiting of P. multocidaantiserum may be masked by other factors present.

EXAMPLE 13 Hatchability of Eggs Injected at Day 18 of Incubation with P.multocida antiserum-P.multocida CFUs

[0089] This study was designed to test the effect of serumantibody-bacterium complexes when administered in ovo to SPF eggs. Thesame number of CFUs (five were targeted) of P. multocida strain M9 wasmixed with varying amounts of P. multocida antiserum and then 0.1 ml ofeach mixture was inoculated into fifteen SPF eggs in each of sevengroups. A 100 CFUs/ml stock solution was prepared using P. multocidastrain M9 culture. To insure that each egg received the same number ofCFUs, the appropriate amount of serum was mixed with the appropriateamount of the stock solution for each group at five minute intervals.These mixtures remained room temperature for thirty minutes, after whichtime, 0.1 ml was inoculated into fifteen eggs at day eighteen ofincubation. The 100 CFUs/ml stock solution was plated in triplicatefollowing the preparation of the final group's mixture and again afterthe final group's mixture reacted for 30 minutes (Table 13). the plateswere all incubated at 37 degrees C. for 24 hours, after which, colonieswere counted. Table 14 provides hatchability data for each group. TABLE13 Titer of the planned 1 × 10(2) P. multocida Strain M9 Stock SolutionCFUs/0.5 ml Average Per 1 ml Pre-30 minutes 38 32 64 28 30 Post-30minutes 30 29 58 30 27

Hatchability effects of inoculating SPF eggs at day 18 of incubationwith 1 ml of mixtures containing antiserum to P. multocida and CFUs ofthe M9 strain of P. multocida.

[0090] TABLE 14 Hatchability effects of inoculating SPF eggs at day 18of incubation with 1 ml of mixtures containing antiserum to P. multocidaand CFUs of the M9 strain of P. multocida. Group 7 Group 2 Group 4 Group5 Group 6 50.0 μl Group 1 0.1 μl Group 3 10.0 μl 25.0 μl 50.0 μl serumno serum serum 1.0 μl serum serum serum serum 50.0 μl PBS +3.2 CFUs +3.2CFUs +3.2 CFUs +3.2 CFUs +3.2 CFUs +3.2 CFUs egg Normal 2 1  2 4  15Hatched Hatched (Dead) Hatched 1 3 3  3 5 (clinically affected)Un-hatched 14 13 11 12 10 6 % 6.7 13.3 26.7 20.0 33 60.0 100 hatched

[0091] This study was originally designed to inoculate each egg that wasto receive bacteria with 5.0 CFUs mixed with the appropriate volume ofP. multocida antiserum. The titer information found in Table 13 showsthat the eggs received a number of CFUs closer to 3.2 than to 5 and thatlittle colony loss occurred as a result of the 30 minute reactionperiod. The results in Table 14 show that even small numbers of CFUs ofP. multocida strain M9 are devastating to SPF eggs when administered atday eighteen of incubation. The controls in group 7 experienced a 100%hatch. The hatch was severely affected in the other groups. Of thesegroups, 5 and 6 contained the next highest percentages of total hatchedbirds. The eggs in group 6 were inoculated with the highest ratio ofantiserum to P. multocida (50 μl+3.2 CFUs) and experienced a 60% overallhatch with a 27% normal hatch. This trend suggests that antiserum to P.multocida, when combined with the live bacteria, may provide some degreeof protection to a chicken embryo by either decreasing or delaying thepathogenic effects of the bacterium.

[0092] The data in Tables 12 and 14, comparing serum with and without P.multocida antibodies and different amounts of serum antibodiesrespectively, show a possible inhibitory effect of serum antibodies toP. multocida on the growth, and perhaps the pathogenic effects, of theorganism. In Table 14 it was shown that the two highest amounts ofantibody (Groups 5 and 6) resulted in better hatches compared to thebacterium alone (Group 1).

EXAMPLE 14 Growth of M. gallisepticum; Production of Hyperimmune Sera

[0093] A culture of the bacterium Mycoplasma gallisepticum strain F wasobtained from North Carolina State University, Mycoplasma Lab, Collegeof Veterinary Medicine. The F strain of M. gallisepticum is used in thecommercial layer industry as a live vaccine. Forty milliliters of Frey'sMedia supplemented with 15% swine serum (FMS) was inoculated with 1.33mls of the bacterial culture. This mixture was then incubated forapproximately 18 hours at 37 degrees C. and the grown culture was mixed80/20 with sterile glycerol for freezing at −70degrees C. Sterility ofthis mixture was tested on Trypticase Soy Agar (TSA) and no extraneousorganisms grew. Titer determination for the M. gallisepticum strain Fstock culture after 24 hours at −70degrees C. was 5.8×10(8) CFUs/ml.

[0094] Antiserum to M. gallisepticum strain R was purchased from theNCSU Mycoplasma Lab. The antiserum was produced by hyperimmunizing NewZealand White rabbits with inactivated M. gallisepticum strain R inadjuvant. Rabbits were immunized by intramuscular and intradermalinjections three times prior to blood collection. This antiserum isdesignated as MGA.

EXAMPLE 15 Growth Inhibition Effect of MGA on M. gallisepticum Strain F

[0095] This experiment investigated the growth of a given amount of M.gallisepticum strain F over time after the organism was mixed withdifferent amounts of MGA. A sample of MGA was initially diluted 1:10 inphosphate buffered saline (PBS). Then, the antiserum was further dilutedby making 10 serial 1:2 dilutions by adding 0.5 ml of the previousdilution to 0.5 ml of PBS (dilutions 1:20 through 1:10240). One vial ofM. gallisepticum F stock culture was thawed at room temperature anddiluted 1:100. This 10(−2) stock solution contained 5.8×10(6) CFUs/ml.Bacterium-antibody complexes were prepared by adding 0.4 ml of the5.8×10(6) stock solution to 0.4 ml of each of the 11 MGA dilutions.These complexes were allowed to react at room temperature for 30minutes. Treatment 12 consisted of 0.4 ml PBS added to 0.4 ml of thesame bacterial stock solution.

[0096] Following the 30 minute reaction time, each of the 12 treatmentswas serially diluted in FMS from 10(−1) through 10(−8). These tubes wereincubated for 14 days and growth was determined at 41 hours, 47.5 hours,and at 14 days. (Growth of M gallisepticum is detected in FMS by a colorchange. As bacterial growth increases the pH of the medium decreases,causing a color change in the pH indicator phenol red. As growth occursthe color gradually changes from a deep red to orange and eventually toyellow. The degree of growth can be scored based on the medium color).Results are provided in Table 15. TABLE 15 Growth of MgF after 0.4 ml of12 antiserum dilutions, containing between .039 μl and 40.0 μlantiserum, were mixed with 0.4 ml of an MgF stock solution Growth after41 Hours Growth after 47.5 Hours Treat- .4 ml 10 10 10 10 10 10 10 10 1010 MgF ment MgF* MgF** (−1) (−2) (−3) (−4) (−5) (−1) (−2) (−3) (−4) (−5)Presence*** 1 40 .4 ml − − − − − − +/− − − − 10(−7) μl 2 20 .4 ml − +/−− − − − ++ + − − 10(−6) μl 3 10 .4 ml +/− +/− +/− − − − +++ + − − 10(−6)μl 4 5 .4 ml ++ +/− − − − ++++ +++ + − − 10(−7) μl 5 2.5 .4 ml ++++ +/−− − − ++++ +++ +/− − − 10(−6) μl 6 1.25 .4 ml ++++ +/− − − − ++++ ++++/− − − 10(−6) μl 7 .625 .4 ml ++++ +/− +/− +/− − ++++ +++ − − − 10(−6)μl 8 .313 .4 ml ++++ + +/− +/− +/− ++++ +++ + +/− +/− 10(−6) μl 9 .156.4 ml ++++ +/− +/− − − ++++ +++ +/− − − 10(−7) μl 10 .078 .4 ml ++++ +/−+/− − − ++++ ++ +/− − − 10(−7) μl 11 .039 .4 ml ++++ +/− +/− − − ++++ +++/− − − 10(−6) μl 12 .4 ml ++++ +/− − − − ++++ ++ − − − 10(−7)

Growth of MgF after 0.4 ml of 12 antiserum dilutions, containing between0.39 μl and 40.0 μl antiserum, were mixed with 0.4 ml of an MgF stocksolution

[0097] Growth was detected in all 12 treatments by day 14 (Table 1).Growth was delayed in Groups 1-4, the groups containing the highestlevels of MGA. Growth in these groups was not evident as early as in thegroups with lower levels of MGA. These findings suggest that the higherlevels of MGA had a growth inhibiting effect on the bacterium.

EXAMPLE 16 Growth Inhibition Effects of MGA on M. gallisepticum

[0098] A vial of M. gallisepticum strain F stock was thawed and diluted1:100 in FMS. This 10(−2) dilution contained approximately 5×10(6)CFUs/ml. One ml of the 10(−2) stock dilution was placed into each ofeight dilution tubes after being thoroughly mixed. A certain amount ofMGA was added to each tube (see Table 16) and the bacterium/antiserumcomplexes were mixed. They were incubated at room temperature for 15minutes and then at 37degrees C. Growth was determined over the courseof 13 days using color change in the FMS growth medium as an indicator.Results are given in Table 16. TABLE 16 μls of Mg MgF Antiserum Growthafter 27 Growth after 46 Growth after 13 days 5.0 × 10(6) CFU/ml (MGA)hours at 37° C. hours at 37° C. at 37° C. 1.0 ml No serum +++ ++++ ++++1.0 ml 48 no growth no growth ++++ 1.0 ml 24 no growth no growth ++++1.0 ml 12 no growth no growth ++++ 1.0 ml 6 no growth + ++++ 1.0 ml 3 nogrowth ++ ++++ 1.0 ml 1.5 no growth +++ ++++ 1.0 ml 0.5 ++ ++++ ++++

[0099] Moderate growth of M gallisepticum occurred within the first 27hours of incubation in the tube that did not contain antiserum andgrowth was heavy in this tube by 46 hours at 37 degrees C. (Table 16).Bacterial growth was not detected within the first 27 hours ofincubation in the tubes that contained greater than or equal to 1.5 μlsMGA. After 46 hours of incubation, growth was still not detected in thetubes that contained 12, 24, and 48 μls of MGA. All tubes showed heavygrowth of M. gallisepticum after 13 days at 37 degrees C. These resultsshow that the bacterium was present in all tubes, but that higheramounts of antiserum delayed growth for longer periods of time than didlesser amounts. The time that it took for growth to be detected appearsto be directly proportional to the amount of MGA in thebacterium-antiserum complex.

EXAMPLE 17 Effect of M. gallisepticum-MGA Complexes on Hatch

[0100] Nine groups of eggs were inoculated at day 18 of incubation.Seven of the groups were inoculated with one of seven different M.gallisepticum strain F-MGA complexes. One group was inoculated with thebacterium only and another group was inoculated with a 1:4 dilution ofFMS in PBS.

[0101] A vial of M. gallisepticum strain F stock was thawed and diluted1:5 and 1:10 in 10% FMS and 90% PBS diluent. Appropriate amounts ofthese dilutions were used to create the bacterium-MGA complexes. Eachegg received a 0.1 ml injection containing the same number of M.gallisepticum CFUs with the appropriate amount of antiserum for aparticular group, except for Group 1. The eggs in Group 1 received 0.1ml of the FMS/PBS diluent. The complexes were allowed to react togetherfor 10 minutes before inoculation. The eggs were then incubated untilhatch.

[0102] The 1:10 dilution of the bacterial stock was titered by making 3serial dilution series through 10(−9) and plating the 10(−6) dilutionsof two dilution series TSA and incubating at 37° C. All tubes in allthree serial dilution series showed M. gallisepticum growth. The titerwas 8×10(8) CFUs/ml. Hatchability results are provided in Table 17.TABLE 17 # Eggs # Healthy Amount of Hatched/ Chicks/ Mg Antiserum # Eggs# Eggs Group MgF CFUs (MGA) Injected Injected 1 0 0 13/14 (93%)  12/14(86%) 2 8.0 × 10(6) 40 μl 10/11 (91%)   8/11 (73%) 3 8.0 × 10(6) 20 μl10/10 (100%)  8/10 (80%) 4 8.0 × 10(6) 10 μl 6/11 (55%) 0/11 (0%) 5 8.0× 10(6) 5 μl 10/10 (100%)  2/10 (20%) 6 8.0 × 10(6) 2 μl 6/11 (55%) 1/11(9%) 7 8.0 × 10(6) 0.5 μl 7/11 (64%) 0/11 (0%) 8 8.0 × 10(6) .05 μl 8/11(73%) 0/11 (0%) 9 8.0 × 10(6) 0 8/14 (57%) 0/14 (0%)

[0103] The MGA-M.gallisepticum complexes influenced the percent hatchand chick health. The groups that experienced hatches above 90% were thegroups that contained the largest proportions of MGA to CFUs (with theexception of Group 4). The percentage of health chicks was much higherin Groups 2 and 3 than in other groups receiving MGA-bacterium complexeswith less antiserum in the formulation. These findings indicate thatcertain MGA:bacterium ratios have the capability of protecting adeveloping chicken embryo by delaying and/or decreasing the pathogeniceffects of the bacterium.

EXAMPLE 18 M. gallisepticum strain F/Antibody Complex Vaccine

[0104] Six groups of 16 viable eggs were inoculated at day 18 ofincubation. The 16 eggs in the negative control group (Group 6) wereinoculated on day 18 with 0.1 ml of diluent (1 part FMS in 9 parts PBS),and hatched in a large hatcher unit that contained no other eggs.

[0105] A vial of M. gallisepticum strain F stock was thawed at roomtemperature and diluted 1:5 (stock 2). The titration of stock 2 showedthat it contained 7×10(7) CFUs/ml. Stock 2 was then divided into 0.9 mlaliquots and combined with 0,5,10,20 or 40 μl of MGA. Once mixed, thebacterium-MGA formulations were allowed to incubate at room temperaturefor 15 minutes. These bacterium-MGA complexes were administered to eggsof each group in 0.1 ml doses. Group 1 eggs received inoculationscontaining only bacteria. Each group of 16 eggs was then placed inseparate small hatcher units until day of hatch. The MGA-M.gallisepticum formulations tested CFUs are shown in Table 18.

[0106] The remaining stock 2 dilution was titrated in three separateserial 10-fold dilutions to 10(−9) using FMS. The 10(−4), 10(−5) and10(−6) dilution tubes in each series was plated in quadruplicate on FMSagar and incubated at 37° C. for 9 days.

[0107] On the day of hatch, Groups 1-5 were processed. Normal, healthylooking chicks were sampled for the presence of M. gallisepticum byswabbing the choanal cleft with a sterile swab and inoculating tubescontaining 1.8 mls of FMS. After the chicks were processed, the sampledchicks from each group were placed in a P2 containment room. Each groupwas placed in a separate brooder cage and no two cages were in contact.

[0108] The chicks in vehicle control Group 6 experienced a delayed hatchand were processed the day following the hatch of groups 1-5. Tencontrol birds were swabbed for the presence of M. gallisepticum and wereplaced in a brooder cage in a separate P2 containment room. On 21 daysof age, all surviving chicks were bled and serum collected fordetermination of antibodies to M. gallisepticum by serum plateagglutination (SPA) and ELISA. Results are provided in Table 18. TABLE18 Group 1* 2 3 4 5 6 #CFUs/egg 1.4 × 10(6) 3.5 × 10(6) 3.5 × 10(6) 3.5× 10(6) 3.5 × 10(6) no CFUs MGA/egg 0 μl 5 μl 10 μl 20 μl 40 μl 100 μldiluen # Normal 0 0 0 8 12 10 Hatched # Clinically 9 7 8 3 2 1 AffectedHatched Unhatched/ 7 9 8 5 2 5 Dead % Hatched 56.25 43.75 50.0 68.7587.5 68.75 % Normal 0 0 0 50 75 62.5 Hatched # Chicks 0 0 3 9 10 10Placed # Chicks NA NA 1 2 7 10 Alive at 3 weeks MgF Not Tested 3/3 3/39/9 9/10 0/10 Reisolation # Positive/ # Sampled SPA** NA NA 1/1 2/2 6/70/10 # Positive/ # Sampled Mean ELISA NA NA 599 643 645 0 Titer

[0109] The eggs receiving the bacterium without antiserum (Group 1) andthe two lower amounts of MGA plus M. gallisepticum (Groups 2 and 3) hadthe lowest percent hatches and no normal birds hatched. The vehiclecontrol group (Group 6) experienced a delayed hatch as well as a poorerhatch than expected. This probably was caused by the fact that the eggswere incubated in a large hatcher intended for the incubation of 2000eggs. Despite this hatch problem, 10 chicks were healthy and they testednegative for M gallisepticum isolation and the two serum antibody tests.Groups 4 and 5 experienced percent hatches and percent normal hatchesthat showed great improvement over those of Groups 1, 2 and 3. The eggsin these two groups received higher levels of MGA and Group 5(3.5×10(6)CFU+40 μl MGA) experienced the best hatch of all the groups.All birds remaining alive at the time of serum collection were healthy.The antibody response to M. gallisepticum measured by the SPA test andELISA indicate that the M. gallisepticum strain F complex vaccines wereefficacious for birds in Groups 3, 4 and 5.

[0110] It is interesting to note that one bird in Group 5 was negativefor M. gallisepticum reisolation at hatch, the SPA test, and ELISA. Allthe other birds that were sampled from Groups 3,4 and 5 tested positivein each case. It appears that one bird in Group 5 never became infected.

[0111] Examples 14-18 were designed to test the usefulness of abacteria:antibody vaccine complex. The data support the concept thataddition of specific antiserum (specific for the vaccine bacteria) tolive bacteria in the appropriate ratio provides protection to the chickembryo by decreasing or delaying the pathogenic effects of the bacteriumwhile at the same time allowing an efficacious immune response todevelop in the hatchlings, as evidenced by an active humoral immuneresponse.

That which is claimed is:
 1. A method of producing active immunityagainst a bacterial disease in a subject, said method comprising:administering to the subject a vaccine conjugate comprising a livebacteria and a neutralizing factor bound to the live bacteria; theneutralizing factor selected from the group consisting of antibodies andantibody fragments; the antibody or antibody fragment capable ofneutralizing the live bacteria; the vaccine conjugate administered in anamount effective to produce an immune response to the live bacteria inthe subject.
 2. A method according to claim 1, wherein the live bacteriais capable of causing disease in the subject.
 3. A method according toclaim 1, wherein the neutralizing factor is selected from the groupconsisting of IgG immunoglobulins and IgG immunoglobulin fragments.
 4. Amethod according to claim 1, wherein the subject is a bird and saidadministering step is carried out in ovo.
 5. A vaccine preparationuseful for producing active immunity against a bacterial disease in asubject, said vaccine preparation comprising: a pharmaceuticallyacceptable formulation comprising a vaccine conjugate; said vaccineconjugate comprising a live bacteria and a neutralizing factor bound tosaid live bacteria; said neutralizing factor selected from the groupconsisting of antibodies and antibody fragments; said antibody orantibody fragment capable of neutralizing said live bacteria; and saidvaccine conjugate included in said pharmaceutically acceptableformulation in an amount effective to produce an immune response to saidlive bacteria in said subject.
 6. A vaccine preparation as claimed inclaim 5, wherein said live bacteria is capable of causing disease insaid subject.
 7. A vaccine preparation as claimed in claim 5, whereinsaid pharmaceutically acceptable formulation is lyophylized.
 8. A methodof producing active immunity against a protozoal disease in a subject,said method comprising: administering to the subject a vaccine conjugatecomprising a live protozoa and a neutralizing factor bound to the liveprotozoa; the neutralizing factor selected from the group consisting ofantibodies and antibody fragments; the antibody or antibody fragmentcapable of neutralizing the live protozoa; the vaccine conjugateadministered in an amount effective to produce an immune response to thelive protozoa in the subject.
 9. A method according to claim 8, whereinthe live protozoa is capable of causing disease in the subject.
 10. Amethod according to claim 8, wherein the live protozoa is an avianprotozoa and the subject is a bird.
 11. A method according to claim 10,wherein the live protozoa is an Eimeria species.
 12. A method accordingto claim 10, wherein the live protozoa is E. tenella or E. acervulina.13. A method according to claim 8, wherein the neutralizing factor isselected from the group consisting of IgG immunoglobulins and IgGimmunoglobulin fragments.
 14. A method according to claim 8, wherein theneutralizing factor is of polyclonal origin.
 15. A method according toclaim 8, wherein the neutralizing factor is of monoclonal origin.
 16. Amethod according to claim 8, wherein the subject is administered thevaccine conjugate by a method selected from the group consisting ofsubcutaneous adminstration, intraperitoneal adminsitration, andintramuscular administration.
 17. A method according to claim 8, whereinthe subject is a bird and said administering step is carried out in ovo.18. A vaccine preparation useful for producing active immunity against aprotozoal disease in a subject, said vaccine preparation comprising: apharmaceutically acceptable formulation comprising a vaccine conjugate;said vaccine conjugate comprising a live protozoa and a neutralizingfactor bound to said live protozoa; said neutralizing factor selectedfrom the group consisting of antibodies and antibody fragments; saidantibody or antibody fragment capable of neutralizing said liveprotozoa; and said vaccine conjugate included in said pharmaceuticallyacceptable formulation in an amount effective to produce an immuneresponse to said live protozoa in said subject.
 19. A vaccinepreparation as claimed in claim 22, wherein said live protozoa iscapable of causing disease in said subject.
 20. A vaccine preparation asclaimed in claim 22, wherein said pharmaceutically acceptableformulation is lyophylized.
 21. A vaccine preparation as claimed inclaim 22, wherein said live protozoa is an avian protozoa.
 22. A vaccinepreparation as claimed in claim 22, wherein said live protozoa is anEimeria species.
 23. A vaccine preparation as claimed in claim 22,wherein said live protozoa is E. tenella or E. acervulina.
 24. A methodof producing active immunity against a mycoplasma in a subject,comprising: administering to the subject a vaccine conjugate comprisinga live mycoplasma and a neutralizing factor bound to said mycoplasma,where the neutralizing factor is selected from the group consisting ofantibodies and antibody fragments, said antibody or antibody fragmentcapable of neutralizing said live mycoplasma; the vaccine conjugateadministered in an amount effective to produce an immune response to thelive mycoplasma in the subject.
 25. A method according to claim 24wherein said mycoplasma is Mycoplasma gallisepticum.
 26. A methodaccording to claim 24, wherein the neutralizing factor is selected fromthe group consisting of IgG immunoglobulins and IgG immunoglobulinfragments.
 27. A method according to claim 24, wherein the subject is abird and said administering step is carried out in ovo.